Immune stimulating and controlling composition comprising bacterial chromosomal dna fragments and non-toxic lipopolysaccharides

ABSTRACT

The present invention relates to immune stimulating and controlling composition comprising bacterial chromosomal DNA fragments and non-toxic lipopolysacchararides. The composition of the present invention can be industrially applied the effective materials for treating cancers and adjuvant.

TECHNICAL FIELD

The present invention relates to immune stimulating and controllingcomposition comprising bacterial chromosomal DNA fragments and non-toxiclipopolysaccharides.

BACKGROUND ART

Cancer therapy developed from the 1960s has largely involved the use ofsurgery, radio therapeutics and chemotherapy. These treatments haveshown the effect that the upward curve of cancer death rate soared up to1973 in the U.S. becomes sluggish. However, surgery and radiotherapeutics are topical treatment and so they have limitation thatpatients are convalescing favorably only when cancer is early blocked aslocal cancer. Chemotherapy is successful only if all cancer cells arecompletely eliminated and so chemotherapy may damage the host, normaltissue such as immune system of patients and threaten life of the oldand the weak. The main purpose of immuno-therapy is to resist thecancerization by reinforcing immune surveillance. There are severaltrials as follows.

1) Immunological prevention; An animal of the same class was inoculatedwith cancer tissue to prevent homologous cancer. For example, viralleukemia of animal may be prevented using its cause virus (Morton et al.1991, proc. am. assoc. cancer res. 2: 492: 494). However, this methodhas never been applied to a person and it is difficult to inducecellular immunity.

2) Immunotherapy;

Active Specific Immunization

This immunization is to prevent cancer cells activating specific immunecancer supervisory cells by inoculating patients with self-cancer cellsor homologous cancer cells or inactivated self- or iso-cancer cellsregulated by X-ray irradiation or mitomycin-C. However, this methodsucceeded in animal experiment not in people. Recently, in order toenhance the expression of specific antigens in cancer tissue, variousmethods have been of attaching with Con-A or exposing hidden antigens bytreating with neuramindase or of forming hybridoma with heterologouscells. However, the use of dendritic cells (Sprinzl et al, Cancer TreatRev. 2001 August; 27(4): 247-55) or development of other various DNAvaccine treatments (Pantuck et al, Int J Urol. 2001 July; 8(7): S1-4)still have a limit in their safety and effect.

Non-Specific Immunotherapy

This immunization most spotlighted at present is used solely or withchemotherapeutic agents for treating almost all kinds of tumors. Thenon-specific immunotherapy means that it will not be restricted by kindsof cancer. Although various theories on its mechanism have beensuggested, they are on study only it is suggested that the non-specificimmunotherapy stimulate reticuloendothelial system specifically activityof lymphocytes. There is Corynebacterium as the chief material actuallyused in clinical tests. Picibanil (OK-432), which has been used forpatients in Korea already, has been studied and produced mainly inJapanese pharmaceutical company. It has been marketed in Japan, Korea orSoutheast Asia. Materials formed of Picibanil has been used in treatingcancer long before. In 1968, Bush Fehleison et al., Germans, discoveredthat the progress of cancer ceased or previously existing cancerdecreased. In 1891, Coley, surgeon in Chicago, the U.S., made mixedtoxin formed of materials extracted from culture medium of streptococci,which was used for many patients.

BCG (or Tubercle Bacillus) and Associated Material Thereof

Living BCG organism: In the 1960s, Old in the U.S. and Mathe in Francereported that animal cancer could be cured by inoculating BCG. In 1970,Morton in the U.S. reported that melanoma could also be cured byinoculating BCG. As a result, BCG and its associated materials werebroadly used as non-specific immunotherapy. A great amount of BCGinoculation is required to expect increasing immune response. BCG can beinoculated under the skin, directly in cancer tissue region or orallyadministrated. However, the oral administration of BCG is not effectivefor people who were inoculated with BCG in their neonatal days but cameinto contact with tubercle bacillus thereafter (BCG or tubercle bacillusare not absorbed in people having tuberculin positive). In the treatmentusing living BCG organism, there are side effects such as requiring thegreat amount of living BCG organism and ulcer around injection, systemicsymptom like chill, fever or liver function disorder. However, in caseof using the small amount to decrease the side effects, the efficacy isreduced or weak.

Unmethylated CpG DNA

Mammalian DNA is different from bacterial DNA in that they have many CpGinhibitions and cytosine of CpG dinucleotide is selectively methylated.Recently, it has been recognized that CpG motifs in bacterial DNArapidly stimulated the polyclonal B-cells and so increased IgMsecretion, and stopped the progress of cell cycle by anti-IgM antibodyand powerfully inhibited the induction of apoptosis to inhibit c-mycexpression and made myn, blc2 and bcl-XL mRNA expression increase toprotect cells from apoptosis. In other study, it was reported that CpGmotifs activated directly B-cells to increase IL-6 and IL-12 secretionwithin a short time. Clinical test on immune adjuvants and asthmatictreatments using synthesis oligonucleotides including CpG sequences isgoing in progress the CPG Company in the America.

As described above, although treatments have been developed usingdiverse immune regulating materials, BCG and CpG among those treatmentsare just applied to people. Despite broad effects of BCG, it isdifficult to apply a great amount of BCG or by blood injection becauseof its stability. In case of CpG, synthetic oligonucleotides are tooexpensive.

DETAILED DESCRIPTION OF THE INVENTION

Accordingly, the object of the present invention is to provide materialsfor inducing immune response more stable, economic, effective andspecific than the conventional ones.

There is provided immune stimulating and controlling compositioncomprising: bacterial chromosomal DNA fragments; and non-toxic bacteriallipopolysacchrides.

It is preferable that the bacterial chromosomal DNA fragments have sizeranging from 2.0 to 0.5 kb and the lipopolysaccharides have daltonranging from 3,000 to 10,000 dalton. The bacterial DNA sequence includesnot only unmodified but also modified bases like methylated base(cytosine, adenine or guanine).

It is preferable that the least amount of the bacterial chromosomal DNAfragments and the lipopolysaccharides may be mixed to show the effect ofthe present invention. Particularly, the present invention shows theincrease of dose dependent efficacy in a mass ratio ranging from 500:1to 1:500. In the above-described mass ratio, the present invention isnon-toxic and economic.

It is preferable that the bacterial chromosomal DNA fragments and thelipopolysaccharides are mixed by shaking.

The composition of the present invention is useful for immune adjuvantsor anti-cancer treatments. These effects are shown by inducing immuneactivation of T-helper 1 type.

It is preferable that the bacteria in the present invention isEscherichia coli or mycobacteria. More preferably, the bacteria isEscherichia coli, particularly, E. coli EG0021(KCCM-10374).

In the composition of the present invention, synergy effect by CIA02 maybe expected in stability, cell immune induction, synergy effect by CIA05may be expected in immune reinforcement specifically cancer treatment.

The disclosed immune stimulating and controlling composition comprisingbacterial chromosomal DNA fragments and non-toxic lipopolysaccharideswill be described briefly.

The present inventors succeeded in effective production of bacterialoligonucleotides as anticancer adjuvant and development of modifiedlipopolysaccharides for suitable activation as anti-cancer treatments. Anew immune adjuvant, CIA07, is finally obtained by combining thebacterial oligonucleotides and the lipopolysaccharides.

Generally, the combination of lipopolysaccharide and DNA shows synergyeffect. Lipopolysaccharide shows various responses such as serving asindependent antigen of T-cells. Here, the synergy effect may causecrucial results such as sepsis.

The present inventors obtained a strain, E. coli EG0021, having shortcarbohydrate chained lipopolysaccharide from Escherichia coli in healthyhuman intestines. They deposited the strain with No. KCCM 10374 in Koreaculture center of microorganisms, KCCM, located in 361-221 Hongje-dong,Seodaemun-gu, Seoul, Korea, in May 2^(nd), 2002. They established amethod of purifying lipopolysaccharide from this strain.

E. coli DNA, CIA02, representing-immune activation was isolated fromgenomic DNA of E. coli EG0021. The CIA02 was obtained afterfragmentation of the isolated DNA and general treatment.

CIA07 was finally obtained by combination of the CIA02 and the CIA05.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a picture illustrating E. coli chromosomal DNA divided intoeach fraction by using ultrasonicatior to detect the size of E. coli DNArepresenting the optimal effect, wherein Lane 1 represents intact, Lane2 over 1 kb, Lane 3 2-0.5 kb and Lane 4 less 0.5 kb of DNA.

FIGS. 2 a to 2 c are graphs illustrating the optimal immune increasingeffect in E. coli DNA (CIA02) of about 2-0.5 kb.

FIG. 3 is a picture illustrating lipopolysaccharide product isolatedfrom E. coli-outermembrane. The picture illustrates isolatedlipopolysaccharide according to 5 times batch.

FIG. 4 is a picture illustrating that the size of isolated E. colilipopolysaccharide treated with alkali is changed by degrading lipid Aand lose toxicity by this treatment, wherein Lane 1 represents isolatedlipopolysaccharide product CIA04 and Lane 2 alkali-treated non-toxiclipopolysaccharide CIA05.

FIG. 5 is a graph illustrating the decrease of TNF-α secretion in THP-1cell line treated with the non-toxic lipopolysaccharide (CIA05).

FIG. 6 a is a graph illustrating results of general safety test on thenon-toxic lipopolysaccharide (CIA05) in mouse.

FIG. 6 b is a graph illustrating results of general safety test on thenon-toxic lipopolysacchride (CIA05) in guinea pig.

FIGS. 7 a to 7 c are graphs illustrating immune increase effectaccording to combination method and concentration of E. coli DNA (CIA02)and non-toxic lipopolysaccharide (CIA05).

FIGS. 8 a to 8 c are graphs illustrating immune increase effects of E.coli derived CIA07 in comparison with other immune adjuvants.

FIG. 9 is a graph illustrating cytokine secretion in human whole bloodtreated with E. coli derived anti-cancer treatment CIA07.

FIGS. 10 a to 10 b are graphs illustrating the amount of cytokinesecretion in human whole blood treated with E. coli derived anti-cancertreatment CIA07 and mycobacterium DNA.

FIGS. 11 a to 11 b are graphs illustrating the increase of promoteractivity by NF-kB activation after treating E. coli derived anti-cancertreatment CIA07 in raw cells.

FIG. 12 is a graph illustrating inhibition of cancer growth when E. coliderived anti-cancer treatment CIA07 is administrated in C3H/HeJ mouse towhich mouse bladder cancer cell line (MBT2) was transplanted.

PREFERRED EMBODIMENTS OF THE PRESENT INVENTION

The disclosed immune stimulating and controlling composition comprisingbacterial chromosomal DNA fragments and non-toxic lipopolysaccharideswill be described in more details referring to examples below, when arenot intended to be limiting.

EXAMPLE 1 Obtainment of Non-Toxic Strain

Screening and Isolating Mutant E. coli Having Short Carbohydrate ChainedLipopolysaccharide

E. coli EG0021 having short carbohydrate chained lipopolysaccharide wasisolated from healthy human intestines, and a method of purifyinglipopolysaccharide from the strain was estabilished.

A procedure was 5 times repeated of injecting liquid-cultured singlecolony of E. coli isolated from healthy adult male intestines, intoexperimental animal, Balb/C mouse.

50 kinds of strains were selected therein, and one colony in theselected 50 strains was obtained from a plate. After the colony wasdissolved in 4 ml of 0.9% physiological saline solution, 1 ml of thesolution was moved into an eppendorf tube. The solution was treated with2 ul of DNase I and reacted at an incubator at 37° C. for 1 hour. Aftertreatment of DNase the solution was treated with 50 ul of Rnase (10mg/ml) and reacted at an incubator of 37° C. for 1 hour. Then, 10 ul ofProteinase K(20 mg/ml) was put therein and reacted at 37° C. overnight.Human lymphocyte cell line differentiated by GM-CSF was treated with LPSof each strain obtained therefrom. TNF-α secretion was measured and astrain having the least value was selected (see Table 1) and confirmedthe molecular weight of lipopolysaccharide by electrophoresis. It wasshown that the attenuated strain was not morphologically changed or inits characteristics. Lipopolysaccharides having a molecular weightranging from 5,000 to 10,000 without lipopolysaccharide ladder having amolecular weight ranging from 50,000 to 100,000 were shown inelectrophoresis (see FIG. 1). This strain was called EG0021. TABLE 1TNF-α secretion value of E. coli homogenate obtained from healthy humanintestines TNF-a No. (pg/1 ul) EG0001 more (>100) EG0002 12 EG0003 72EG0004 85 EG0005 25 EG0006 35 EG0007 71 EG0008 28 EG0009 2 EG0010 13EG0011 39 EG0012 64 EG0013 8.8 EG0014 9 EG0015 70 EG0016 more (>100)EG0017 6 EG0018 11 EG0019 0.3 EG0020 80 EG0021 0.1 EG0022 more (>100)EG0023 more (>100) EG0024 more (>100) EG0025 53 EG0026 12 EG0027 4EG0028 76 EG0029 92 EG0030 more (>100) EG0031 21 EG0032 1.2 EG0033 more(>100) EG0034 more (>100) EG0035 7 EG0036 87 EG0037 0.7 EG0038 39 EG003937 EG0040 91 EG0041 65 EG0042 54 EG0043 More (>100) EG0044 More (>100)EG0045 17 EG0046 2.1 EG0047 3.5 EG0048 More (>100) EG0049 More (>100)EG0050 More (>100)

EXAMPLE 2 E. coli DNA Preparation Method

E. coli Chromosomal DNA Purification

E. coli EG0021 was cultured by shaking in TSB (Tryptic soy broth; Difco)culture medium (30 g/L) at 37° C. for 10 hours.

After 10 L cultivation, 150 g of cells obtained by centrifugtion at8,000 G was washed in TE(10 mM Tris, pH 8.0, 25 mM EDTA) buffer solution(300 ml) and centrifuged. The cells (150 g) obtained by centrifugationwas dissolved in 750 ml of lysis solution (10 mM Tris (pH 8.0), 25 mMEDTA, 100 ug/mL Lysozyme) and treated at 37° C. for 1 hour.

Thereafter, proteinase K (Sigma) was added in the solution to finalconcentration 100 ug/ml, and treated at 50° C. for 12 hours.

Mixing the solution with penol/chloroform/isoamyl alcohol (25:24:1) at aratio of 1:1 was repeated three times to obtain water layer.

E. coli chromosomal DNA was obtained by ethanol precipitation.

After purified E. coli DNA was diluted using sterile distilled water,the concentration of the E. coli DNA was measured at 260 nm and 280 nmswith UV spectrometer.

The concentration was measured according to the following method:Double  stranded  DNA  concentration  (ug/ml) = OD260nm × dilution  rate × 50Single  stranded  DNA  concentration  (ug/ml) = OD260nm × dilution  rate × 40OD260nm/OD280nm = 1.7 ∼ 1.8

E. coli DNA Fragmentation

The purified E. coli chromosomal DNA was dissolved in TE buffer solutionto 0.5 mg/ml and sonicated in a glass beaker with ultrasonicator.

-   -   20 ml of the solution was fragmented at one time using 500 watt        sonication VCX500(Sonics Co.) as ultrasonicator and 630-0220        (tip diameter: ½″(13 mm)) as tip.    -   Here, in order to identify the size of E. coli DNA representing        the optimal: effect, the whole E. coli chromosomal DNA was        divided in 20,000 J according to time period using        ultrasonicator and then separated by size (see FIG. 1). E. coli        DNA was divided into the whole DNA before sonication (Intact,        more than 10 kb), 2.0-0.5 kb, 0.5-00.1 kb and less than 00.1 kb        according to its size.    -   In order to identify immune increase effects of E. coli DNA        separated according to size, the effect as immune adjuvant was        measured in mouse (see FIG. 2). 50 ug of HEL (Sigma) as antigen        and 50 ug of each E. coli DNA as adjuvant were injected (i.p)        into ICR mouse (a 4-week old male, 20 g) twice at interval of a        week. 7 days after final injection, the whole blood was        collected and serum was separated. The antibody value in serum        was measured with HEL as antigen using ELISA method (see FIG. 2        a).

As analysis results, the size of 2.0-0.5 kb showed the highest antibodyvalue. Thereafter, from repeated experiments, it was shown that about 1kb represented the optimal effect.

The effects of humoral and cellular immunity in the subclass of antibodyin serum was identified with the same ELISA method (see FIGS. 2 b and 2c).

The sonication condition for obtaining 1 kb E. coli DNA determinedaccording to the above result is at 7 minutes in 20,000 J.

EXAMPLE 3 Removal of Endotoxin from E. Coli DNA and Measurement of DNAPurity

Removal of Endotoxin

After sonication, DNA was reacted with chloroform at 4° C. for 12 hours,and three volumes of ethanol was treated therein to obtain aprecipitate.

The precipitate was treated with Triton X-114 (Sigma) to 0.5% of finalconcentration. The resulting precipitate was reacted at 4° C. for 4hours, warmed at 37° C. for 5 minutes and then mixed withpenol/chloroform/isoamyl alcohol (25:24:1) at a ratio of 1:1 to obtainwater layer.

The obtained E. coli DNA was precipitated with ethanol and dissolves inpyrogen free water.

Endotoxin removed DNA was analyzed with Limulus Amebocyte Lysate (LAL)kit (BioWhittaker QCL-1000) to detect the remaining endotoxin.

Table 1 shows the endotoxin value and yield of purified E. coli DNA(CIA02) after removal of endotoxin according to the above method. TABLE2 The endotoxin value and yield of purified E. coli DNA(CIA02) Amount ofthe whole Endotoxin Sample DNA DNA Pyrogen (per DNA Number Concentration(/15 ml) free DNA Ratio 1 mg/ml) Yield 1 3 mg/ml 45 mg  16.2 mg 1.77 <1ng 36% 2 20.25 mg 1.66 <1 ng 45% 3  18.9 mg 1.71 <1 ng 42%

The amount of remaining organic solvent was measured with GC/MSD (gaschromatography/mass selected detector), HP-5890A/HP-5870B. Ethanol,acetone, chloroform and penol were measured with SIM (Selected IonMonitoring) having the column of 50 m.ultra-1 (see Table 2). TABLE 3Amount of remaining organic solvent Remaining organic solvent AcetoneEthanol Phenol Chloroform ng/ul — 0.813 — —

-   -   More than 99% degree of purity was identified by measuring        protein contamination per E. coli DNA mg with Brad-Ford method.

EXAMPLE 4 Purification of Lipopolysaccharide (CIA04) from Mutant E. Coli

Purification of Lipopolysaccharide from Mutant E. Coli

E. coli was prepared with the same method as above described DNAisolation method.

The prepared E. coli was mixed with 2 volumes of ethanol thereof, andcentrifuged at 4,000 g to obtain a precipitate. 1.5 volumes of acetoneof the precipitate was added, mixed and then centrifuged at 4,000 g.

The same amount of ethyl ether was added and mixed in the resultingprecipitate, and then centrifuged at 4,000 g. The cell pellet obtainedtherefrom was covered with aluminum foil and punctured the foil anddried to measure cell mass. Thereafter, 7.5 ml of extraction mixture(90% penol:chloroform:petroleum ether=2:5:8) was added per 1 g ofcellular dry weight.

The resulting solution was divided into glass centrifuge tube andcentrifuged at 25° C.; 3,000 rpm (1,200 g) for 20 minutes to obtainsupernatant. The supernatant was left in hood for 12 hours. Then, thesolution was divided into glass centrifuge tube and lipopolysaccharidesdissolves in ethyl ether by centrifugation at 25° C., 3,000 rpm (1,200g) for 20 minutes, and then transferred into eppendorf tube. Thesolution was dried in hood, and dried weight was measured with chemicalbalance. Then, ethanol was added therein and stored before use.

After ethanol was completely eliminated in purified E. colilipopolysaccahride stored in ethanol, the amount of KDO(2-Keto-3-deoxyoctonate) in lipopolysacchardie was measured withlipopolysaccharide standard (Lsit Biological Lab.). After theconcentration was measured from the standard, the lipopolysaccharideswere analyzed with SDS-PAGE according to size and identified by silverstaining (see FIG. 3). The lipopolysaccharide had molecular weightranging from about 5,000 to 10,000, and its size was very small comparedwith general E. coli lipopolysaccharide.

EXAMPLE 5 Removal of Toxicity in Purified Lipopolysaccharide from MutantE. Coli

Removal of Toxicity in Lipopolysaccharide by Lipid A Degradation

Purified E. coli lipopolysaccharides diluted to 3 mg/ml of concentrationand mixed with 0.2N NaOH at a ratio of 1:1. The resulting solution wasshaken every 10 minutes at 60° C. and deacylated for 140 minutes.

-   -   About 1/5 volumes of initial 0.2N NaOH of 1N acetic acid was        added in the resulting solution to titrate pH 7.0.    -   After pH titration, ethanol-precipitated non-toxic        lipopolysaccharide was obtained.    -   After the concentration of non-toxic lipopolysaccharide was        measured with KDO method, its size change was identified by        SDS-PAGE and silver staining in comparison with        lipopolysaccharide before treatment.    -   As a result of staining, it was shown that lipid A of        lipopolysaccharide was degraded by alkali treatment and the size        of lipopolysaccharide became smaller (see FIG. 4).

Confirmation of Toxicity Removal of Non-Toxic Lipopolysaccharide

In order to test stability of non-toxic lipopolysaccharide, experimentson secretion, pyrogenicity and abnormal toxicity of inflammatoryproteins were performed.

Experiment on Secretion of Inflammatory Protein

THP-1 (Acute monocytic leukemia) was treated with non-toxiclipopolysaccharide from high to low concentration to measure the amountof secreted TNF-α in comparison with the control group of purifiedlipopolysaccharide.

While 5 pg TNF-α was secreted in 1 ug of lipopolysaccharide in thecontrol group, 0.1 pg TNF-α was secreted in 1 ug of non-toxiclipopolysaccharide. Here, it was shown that inflammatory reactioninduced by toxicity decreased by 50 times. Additionally, it was shownthat the amount of TNF-α secreted in E. coli DNA was below 100 fg. As aresult, the non-toxic lipopolysaccharide was proved to be very safematerial (see FIG. 5).

Experiment on General Safety Test

The sample of high dose was injected in more than two kinds of rodentsto observe abnormal weight change.

A. Experiment in Guinea Pig

About 350 g of a guinea pig showed no abnormality and gained weightgradually when observed for more than 5 days before use.

The 5 ml of sample was used per one guinea pig.

The sample was one time injected (i.p) into more than two guinea pigs,and they were observed for more than 5 days.

B. Experiment in Mouse

An about 5-week old mouse showed no abnormality and gained weightgradually when observed for more than 5 days before use.

The sample was one time injected (i.p) into more than two mice, and theywere observed for more than 7 days.

The sample was proved suitable in this experiment when an animal showedno abnormality during the observation period.

As an experimental result, no abnormal weight change was observed afterinjection of the sample (see FIG. 6 a).

Pyrogenicity Experiment

After vaccine was injected into three rabbits, change in the rectaltemperature was observed. The 0.2 ug/ml of sample per 1 kg of rabbit wasinjected in ear vein of rabbit. Then, the change in abnormal temperaturewas measured by inserting a thermometer into the rectum.

Here, the weight of rabbits was over 1.5 kg. The rabbits were reusedmore than 3 days after they had been used in experiments. The bodytemperature was measured with an apparatus measuring the temperature upto 0.1° C. An injector and its needle were heat-sterilized at 250° C.for over 30 minutes. Only water was fed from 16 hours before use tocompletion of the experiment. The animals were fixed not as tight aspossible.

The body temperature was measured by inserting the thermometer into therectum to a constant depth ranging from 60 to 90 mm for constant time.The temperature measured before injection was defined as a controltemperature. The sample heated at 37° C. was injected into the ear veinwithin about 15 minutes after the control was measured. The bodytemperature was measured every 3 hours, at least 1 hour, afterinjection. Gap between the control temperature and sample temperaturewas defined as difference in temperature. The maximum value of thedifference in temperature was defined as pyrogen reaction of theexperimental animals. Here, the samples of three animals were used.

Pyrogenic material experiment was negative when the total of threeanimals was below 1.3° C. while positive when over 2.5° C. Theseexperiments were performed three times, and the negative reaction wassuitable for these pyrogenic material experiments.

The results are shown in Table 4. TABLE 4 The Before injection Sum ofNo. (three times After injection increase of measured) (hrs) increasedbody time Number 1 2 3 0.5 1 1.5 2 2.5 3 bod Temp. Temp. Result standard1 1 39.1 39.2 39.2 39.4 39.3 39.2 39.2 39.1 39.1 0.2 0.8 pass <1.3° C. 239 39.1 39.3 39 39.2 39.5 39.2 39.1 39.3 0.4 3 39.4 39.2 39.2 39.3 39.539.3 39.5 39.3 39.4 0.2 2 1 39 39.3 39.1 39.4 39.2 39.3 39.1 39.2 39 0.41.7 pass <3.0° C. 2 39.4 39.2 39.2 39.1 39.3 39.1 39.2 39.2 0.3 3 39.339.3 39.2 39.4 39.4 39.4 39.4 39.3 0.2 3 1 39.2 39.2 39.1 39.2 39.2 3939.2 39.1 39.1 0.2 2.5 pass <5.0° C. 2 39.1 39.5 39 39 39.1 39.2 39.139.3 39.2 0.4 3 39.2 39.3 39.2 39.3 39.2 39.3 39.2 39.4 39.3 0.2

EXAMPLE 6 Combine E. Coli DNA Fragment (CIA02) and Non-ToxicLipopolysaccharide (CIA05) and Identification of Activity

Combine E. coli DNA Fragment (CIA02) and Non-Toxic Lipopolysaccharide(CIA05)

The E. coli DNA (CIA02) and non-toxic lipopolysaccharide (CIA05)prepared according to each standard method was mixed to determine theoptimal mixing method and dose.

In order to identify the change in effect according to mixing method,the antigen was treated with two different chemical materials (MPBH andSPB) for binding. CIA02 and CIA05 were combined with the modifiedantigen. In another way, CIA02, CIA05 and the antigen were mixed andshaken. Additionally, immune increase effects in each dose wereanalyzed. In order to identify the effect of immune adjuvant in mouse,0.1 ml of 50 ug HEL (Sigma) as antigen was injected (i.P) into ICR mouse(a 4-week old male, 20 g) two times at the interval of a week. 7 daysafter final injection, the whole blood was collected and serum wasseparated therefrom. The antibody value in serum was analyzed using HELas antigen by ELISA method (see FIG. 7 a).

The control group was 0.5 and 1 ug of LPS(CIA04) while the experimentalgroup was antigen, 0.5 ug of CIA05 and 50 ug of CIA, shaking mixinggroup consisting of antigen, 1 ug of CIA05 and 50 ug of CIA, modifiedantigen, 0.5 ug of CIA05 and 50 ug of CIA, combining group consisting ofmodified antigen, 1 ug of CIA05 and 50 ug of CIA respectively.

As an experimental result, it was proved that LPS(CIA04) represented thehighest antibody activity but it was not suitable for anti-canceradjuvant because of side effects of toxicity (see FIG. 7 a). Althoughcombining methods made little difference, a shaking mixing method wasmore superior to the other methods. Particularly, it was shown that theshaking mixing method was effective because it showed a difference inIgG2a related to cellular immunity among immunoglobulin subclass (seeFIG. 7 b). In yield, the shaking mixing method was simple and had noloss during treating processes. As a result, the shaking mixing methodwas selected. The dose was determined according to the amount of CIA05.The dose of 1 ug showed more superior efficacy than that of 0.5 ug. As aresult, the dose of 1 ug was determined.

The effects of humoral and cellular immunity in subclass of antibody inserum were analyzed with ELISA method.

Experiment Compared with the Conventional Immune Adjuvant

In order to test immune increase effects of the determined making methodand dose, an experiment was performed in comparison with those of theconventional immune adjuvant (see FIG. 8 a).

Applicability for CIA07 as immune adjuvant was analyzed through animalexperiments. 0.1 ml of 50 ug HEL (Sigma L-6876) as antigen was injected(i.P) into ICR mouse (a 4-week male, 20 g) twice at the intervals of aweek. 7 days after final injection, the whole blood was collected andserum was separated therefrom. The antibody activity in serum wasanalyzed with HEL as antigen using ELISA method.

It was shown that the antibody activity of CIA07 represented the sameeffect of CFA (complete Freund's adjuvant), the conventional immuneadjuvant for animal experiment, and Alum (aluminium hydroxide gel) onlyauthorized in use for human (see FIG. 8 a). However, as a result ofanalyzing isotype switching, it was shown that the conventional immuneadjuvants such as CFA and Alum activated immunization of Th(T helper)-2type wherein antibodies of IgG1 were mainly produced while CIA07 inducedimmune activation of Th(T helper)-1 type wherein antibodies of IgG2arather than IgG1 were specifically produced (see FIGS. 8 b and 8 c).

In order to identify the dose of CIA02, 1 ug of CIA05 was mixed in 25 ugand 50 ug of CIA02, respectively. As an analysis result, it was shownthat the antibody activity was changed according to the dose.Accordingly, 50 ug of CIA02 and 1 ug of CIA05 were determined for theoptimal dose.

Identification Experiment CIA Activity with Whole Blood Analysis

Venous blood from healthy male adult was sterilely obtained in vacuumtube having heparan as anticoagulant. The whole blood obtained therefromwas mixed with RPMI 1640 culture medium (2 mM L-glutamine, 1 mM Sodiumpyruvate, gentamycin of 80 ug/ml) at a ratio of 1:1. 20 ul of CIA07 50ug of CIA02+1 ug or 500 ng, 100 ng of CIA05) or 20 ul of HBSS were addedin 1 ml of the whole blood mixed with culture medium and then incubatedin 5% CO₂ culture medium at 37° C. for 24 hours. The secretion amount ofIFN-γ (R&D system, 210-TA-010) and IL-12 p40(R&D system, 2219-IL-005)was analyzed in supernatant liquid in the culture medium with ELISA kit.The results were shown in FIG. 9.

In the same way, 20 ul of CIA07 (50 ug of CIA02+1 ug of CIA05), CIA02(50 ug), Mycobacterium DNA (50 ug) or HBSS (20 ul) were added in 1 ml ofthe whole blood mixed with culture medium. Then, cytokine secretion wasanalyzed.

As an analysis result, the synergic immune increase effect wasrepresented in case of addition of mixed CIA02 and CIA05 more than incase of addition of only CIA02 or only CIA05. The best result was shownin the dose of 50 ug of CIA02 and 1 ug of CIA05. In comparison withmycobacterium DNA, CIA07 represented the excellent result as well asCIA02.

Luciferase Assay

Raw cells were spread in 12 well plates by 5×10⁴ cells (1 ml DMEM/10%FBS) per a well, and cultured in CO₂ incubator at 37° C. for 24 hours.IL-12 luciferase reporter plasmid (0.2 ug/well) mixed with PRL-nullplasmid (20 ng/well) was added in serum free DMEM (50 ul/well) mixedwith Fugene 6 (1.5 ul/well, Roche Cat. No. 1 814 443), transfectionreagent, and then left for 5 to 10 minutes. The resulting mixture wasadded in the Raw cells by 52 ul/well, and incubated in CO₂ incubator at37° C. for 24 hours. Thereafter, Raw cells were treated with CIA07(CIA02 20 ug+CIA05 400 ng per a well) and incubated in CO₂ incubator at37° C. for 12 hours. Luciferase reaction carried out by luciferase assaykit (Promega Cat. no. E1500) to measure luciferase activity withluminometer.

As a result of measuring luciferase activity to identify effects ofCIA07 on IL-8 and IL-12 promoter activity, NF-kB binding site existed inIL-8 and IL-12 promoters in common. It was shown that NF-kB wasactivated in RAW264.7 cell line by CIA07 to increase activity ofpromoters (see FIG. 11).

Measurement of Anti-Cancer Treatment Effect using Cell Lysis Activity ofCIA

Cancer cell killing activity by CIA07 was measured using ₅₁Cr-release.

Antigen only or with CIA07 was injected under the skin of the bottom ofthe foot of a 5˜8 week old male C3H/HeN mouse.

RPMI-1640 (10 mM HEPES, 100 units/ml penicillin, 100 μg/ml sterptomycin,300 μg/ml glutamine; Gibco Laboratories, Grand Island, N.Y.) was usedfor the basal culture medium for culturing cell lines. Inactivated 10%fetal bovine serum (Gibco Laboratories, Grand Island, N.Y.) heated at56° C. for 30 minutes was added in the basal culture medium. In order tomeasure activity of LAK cells and cancer cell mediated killing activity,Sarcoma 180 and mouse bladder cancer cell line (MBT-2) were used fortarget cells.

In order to prepare reaction cell lines, a rat of the experimental groupwere killed using cervical dislocation. Its spleen was sterilelyisolated and minced on stainless steel wire netting using scissors. Thefragments were ground and filtered using a glass stick with addingphosphate buffered saline. Then, tissue debris was removed passingthrough wire netting. After single cell suspension was identified undermicroscope, cells were washed using the basal culture medium one time.The cells were suspended in 0.84% ammonium chloride solution at 37° C.for 5 minutes to dissolve erythrocyte. The cells were further washedusing the basal culture medium two times and suspended in completeculture medium. The cell suspension was divided into culture flasks andcultured in CO₂ constant temperature and humidity chamber at 37° C. for1 hour. Cells that were not attached to the flasks were obtainedtherefrom, and survival cell number was measured using trypan blue dyeexclusion method. Then, 5×10⁶ cells were obtained using the completeculture medium and survival cell number thereof was measured usingtrypan blue dye exclusion method. Then, 5×10⁶ cell/ml of cell suspensionwere made using the complete culture medium.

Target cell line was cultured and the number of cells was counted. 10⁶cells were obtained and the cells were centrifuged at 300 g, 3 minutes.The supernatant liquid except 0.2˜0.3 ml was removed using Pasteurpipette without damaging precipitated cells. 100 Ci Na₂ ₅₁CrO₄ (1 mlCi/ml, NEZ 030S, NEN, USA) was added and labeled in shaking thermostatat 37° C. for 1 hour. The cells were washed using the basal culture andsurvival cell number thereof was measured using trypan blue dyeexclusion method. The labeled target cells were re-suspended in thecomplete culture medium to 5×10⁴ cell/ml.

The labeled target cells were divided by 0.1 ml to put 5×10³ cells per awell on 96 well fine plate having a round bottom. 0.1 ml of reactionscell was added at a ratio of reaction cell:target cell 100:1. The cellswere cultured in 5% CO₂ constant temperature and humidity chamber at 37°C. for 4 hours. After more than 3 wells per an experiment were made andthe culture for 4 hours was finished, the cells were centrifuged at 500g for 15 minutes. Radioactivity was measured in the 0.1 ml ofsupernatant liquid from each well using gamma counter (Packard, USA).Here, in order to induce the maximum emission, 0.1 ml of 5% triton X-100(Sigma, USA) was added in the control well group. In order to measurenatural emission, the labeled cells were cultured in the completeculture medium having the same dose. The cell toxicity was calculatedaccording to the following formula:

-   -   Cytotoxicity(%)=(ER−SR/MR−SR)×100    -   ER: average count (cpm) of experiment group    -   SR: average count (cpm) target cell cultured in culture medium    -   MR: average count (cpm) of target cell treated with 5% Triton        X-100.

The experimental results were shown in Table 5. LAK cells showed celllysis increase by 8 times in comparison with non-immune cells, and by1.5 times in comparison with BCG injection group. MBT-2 cell line showedcell lysis increase by 5 times in comparison with non-immune cells.These results represent possibility of CIA for anti-cancer treatmentsinstead of BCG resulting in various side effects. TABLE 5 Injection days0 3 7 15 Sarcoma 180 Control group 100 100 100 100 BCG 92 ± 4 110 ± 2632 ± 13 189 ± 4 CIA07 94 ± 7 154 ± 3 802 ± 10 109 ± 7 MBT-2 Controlgroup 100 100 100 100 BCG 103 ± 3   96 ± 7 402 ± 11  98 ± 3 CIA07 97 ± 4121 ± 9 513 ± 13 109 ± 6Experiment for identifying anti-cancer activity in mouse

5×10⁵ MBT2 cells (C3H/He derived bladder cancer cell) weresubcutaneously injected at two positions (left, shoulder, right thigh)of a 5-8 week old male C3H/HeJ mouse. Here, several pricks for injectionshould be avoided in order to generate single cancer tissue. The usedmice were 6 to 10 per an experimental group. From the next day afterinjecting tumor cell, 100 ul of CIA07(50 ug of CIA02+500 ng of CIA05) orphysiological salt solution was injected in cell line injected positionone time everyday for 1 week and every two days for the next 2 weeks.The size of cancer generated in the hypoderm was measured three timesper a week using caliper. The results were shown in FIG. 12. It wasshown that the growth of cancer tissue was inhibited in CIA05 injectiongroup in comparison with physiological salt solution injection group.

Industrial Applicability

The anti-cancer treatment CIA07 of mixing two E. coli derived materialsCIA02 and CIA05 according to the present invention has higher safetythan the conventional treatment and minimizes production cost due tosimplicity of production process. Also, CIA07 induces more effective andspecific immunization due to mixing the two materials. Additionally, thepresent invention is cheaper than CpG due to physical process of DNA andmore effective than BCG.

Accordingly, the E. coli derived anti-cancer treatment CIA07 accordingto the present invention is more significant in industrial applicationfor anti-cancer treatment and immune adjuvant.

1-11. (canceled)
 12. A composition comprising: a) bacterial DNAfragments; and b) a molecular weight of about 3,000 to about 10,000daltons of Escherichia coli (E. coli) lipopolysaccharide.
 13. Thecomposition of claim 12, wherein said bacterial DNA fragments compriseunmethylated CpG sequence.
 14. The composition of claim 12, wherein saidbacterial DNA fragments are 0.5 to 2.0 kb.
 15. The composition of claim12, wherein the weight ratio of a) and b) ranges from 500:1 to 1:500.16. The composition of claim 12, wherein a) and b) are mixed by shaking.17. The composition of clam 12, wherein said E. coli lipopolysaccharidecomprises the core antigen, but not the O-antigen.
 18. The compositionof claim 12, wherein said E. coli lipopolysaccharide has a structure asproduced from E. coli strain EG0021 (KCCM-10374).
 19. The composition ofclaim 12, wherein said composition induces a T helper type 1 immuneresponse.
 20. The composition according to claim 12, wherein said E.coli lipopolysaccharide is detoxified with alkali treatment.
 21. Acomposition according to claim 12 wherein said E. colilipopolysaccharide is produced by alkali treatment of lipopolysaccharidefrom E. coli strain EG0021 (KCCM-10374).
 22. A method for producing animmune-stimulating composition, comprising: combining bacterial DNAfragments with non-toxic lipopolysaccharides, wherein said fragments andsaid non-toxic lipopolysaccharides are combined in a ratio of from about500:1 to about 1:500 by weight.
 23. The method of claim 22, wherein saidnon-toxic lipopolysaccharides are E. coli lipopolysaccharides.
 24. Themethod of claim 22, wherein said non-toxic lipopolysaccharides have amolecular weight of about 3,000 to 10,000 daltons.
 25. The method ofclaim 22, wherein said E. coli is E. coli EG0021 (KCCM-10374).
 26. Themethod of claim 22, wherein said non-toxic lipopolysaccharides arerendered non-toxic with alkali treatment.
 27. The method of claim 22,wherein said alkali treatment comprises contacting said non-toxiclipopolysaccharides with NaOH.
 28. The method of claim 22, wherein saidcombining is performed by shaking.
 29. A method of stimulating theimmune system of a mammal having cancer, comprising administering thecomposition of claim 12 to the mammal in an amount effective tostimulate the immune system of said mammal.
 30. A method of inhibitingthe growth of cancer in a mammal, comprising introducing the compound ofclaim 12 into said mammal in an amount effective to inhibit said growthof cancer in said mammal.
 31. A method of inducing a Th1 response in amammal, comprising introducing the compound of claim 12 into said mammalin an amount effective to induce a Th1 response in said mammal.